A tale of two bioconjugations:pH controlled divergent reactivity of protein a-oxo aldehydes in competing a-oxo-Mannich and catalyst-free aldol ligations

Site-selective chemical methods for protein bioconjugation have revolutionised the fields of cell and chemical biology through the development of novel protein/enzyme probes bearing fluorescent, spectroscopic or even toxic cargos. Herein we report two new methods for the bioconjugation of a-oxo aldehyde handles within proteins using small molecule aniline and/or phenol probes. The ‘a-oxo-Mannich’ and ‘catalyst-free aldol’ ligations both compete for the electrophilic a-oxo aldehyde which displays pH divergent reactivity proceeding through the “Mannich” pathway at acidic pH to afford bifunctionalised bioconjugates, and the “catalyst-free aldol” pathway at neutral pH to afford monofunctionalised bioconjugates. We explore the substrate scope and utility of both these bioconjugations in the construction of neoglycoproteins, in the process formulating a mechanistic rationale for how both pathways intersect with each other at different reaction pH.